Xylanases have been found in at least a hundred different organisms. Xylanases are glycosyl hydrolases which hydrolyse β-1,4-linked xylopyranoside chains. Within the sequence-based classification of glycosyl hydrolase families established by Henrissat and Bairoch (1993), most xylanases are found in families 10 and 11. Common features for family 11 members include high genetic homology, a size of about 20 kDa and a double displacement catalytic mechanism (Tenkanen et al., 1992; Wakarchuk et al., 1994). The families have now been grouped, based on structure similarities, into Clans (Henrissat and Davies, 1995). Family 11 glycosyl hydrolases, which are primarily xylanases, reside in Clan C along with family 12 enzymes, all of which are known to be cellulases.
Xylanases can be often used for important applications such as the bleaching of pulp, modification of textile fibers and in animal feed (e.g., xylanases can aid animal digestion, Prade, 1996). Xylanases are useful for production of human foods as well. For example, xylanase improves the properties of bread dough and the quality of bread. Xylanases can also aid the brewing process by improving filterability of xylan containing beers. Xylanases can be employed in the decomposition of vegetative matter including disposal/use of agricultural waste and waste resulting from processing of agricultural products, including production of fuels or other biobased products/materials from biomass.
Often, however, extreme conditions in these applications, such as high temperature and/or pH, etc, render the xylanases less effective than under normal conditions. During pulp bleaching, for example, material that comes from an alkaline wash stage can have a high temperature, sometimes greater than 80° C., and a high pH, such as a pH greater than 10. Since most xylanases do not function well under those conditions, pulp must be cooled and the alkaline pH neutralized before the normal xylanase can function. Taking some of these steps into account, the process can become more expensive since it must be altered to suit the xylanase.
In another example, xylanases are also useful in animal feed applications. There, the enzymes can face high temperature conditions for a short time (e.g. −0.5-5 min at 95° C. or higher) during feed preparation. Inactivation of the enzyme can occur under these temperature conditions, and the enzymes are rendered useless when needed at a lower temperature such as, for example, ˜37° C.
Xylanases with improved qualities have been found. Several thermostable, alkalophilic and acidophilic xylanases have been found and cloned from thermophilic organisms (Bodie et al., 1995; Fukunaga et al., 1998). However, it is often difficult to produce the enzymes in economically efficient quantities. T. reesei, on the other hand, produces xylanases, which are not as thermostable as xylanases from thermophilic organisms. T. reesei is known to produce different xylanases of which xylanases I and II (XynI and XynII, respectively) are the best characterized (Tenkanen et al., 1992). XynI has a size of 19 kDa, a pI of 5.5 and a pH of between 3 and 4. XynII has a size of 20 kDa, a pI of 9.0 and a pH optimum of 5.0-5.5 (Törrönen and Rouvinen, 1995). These xylanases exhibit a favorable pH profile, specificity and specific activity in a number of applications, and can be produced economically in large-scale production processes.
Efforts have been made to engineer a xylanase with favorable qualities. For example, some have tried to improve the stability of the Bacillus circulans xylanase by adding disulphide bridges which bind the N-terminus of the protein to the C-terminus and the N-terminal part of the α-helix to the neighbouring β-strand (Wakarchuk et al., 1994). Also, Campbell et al. (1995) modified Bacillus circulans xylanase by inter- and intramolecular disulphide bonds in order to increase thermostability. Similarly, the stability of T. reesei xylanase II has been improved by changing the N-terminal region to a respective part of a thermophilic xylanase (Sung et al., 1998). In addition to the improved thermostability, the activity range of the enzyme was broadened to include an alkaline pH. Single point mutations have also been used to increase the stability of Bacillus pumilus xylanase (Arase et al., 1993).
By comparing the structures of thermophilic and mesophilic enzymes much information has been obtained (Vogt et al., 1997). Structural analysis of thermophilic xylanases has also given information about factors influencing the thermostability of xylanases (Gruber et al., 1998; Harris et al., 1997).
Currently, however, there is a need for enzymes, especially xylanases, with improved properties in industrial conditions.